PROJECT SUMMARY/ ABSTRACT Chronic wounds affect approximately 6.5 million patients in the United States. Current standard protocols for wound management do not guarantee healing and focus on maintaining a wound environment that is conducive to passive self-healing. Hence, there is a need to develop alternative treatments that promote active healing and shorten healing time leading to reduced costs. We have previously reported that treatment with low-frequency (20-100 kHz), low-intensity (50-150 mW/cm2) ultrasound (LFLI US) significantly (p<0.03) reduces venous ulcer size in vivo as compared to wounds treated with a sham device. This proposal aims at determining the biological mechanisms by which LFLI US promotes chronic wound healing in vitro. There is evidence that the cause of impaired healing is the dysregulation of macrophage phenotype, especially the defective transition from pro- inflammatory (M1) to pro-healing (M2) macrophages. Our characterization of tissue debrided from chronic wounds has shown that healing chronic wounds contain higher proportions of M1-like than M2- like macrophages. Additionally, the signaling protein Rac2, downstream of integrin and focal adhesion kinase activation, is a key regulator of mechanotransduction in macrophages and facilitates the transition of macrophages from the M1 to M2 phenotype. The proposed study will systematically examine the effects of LFLI ultrasound on macrophage phenotype, using macrophages cultured in three-dimensional (3D) scaffolds. We hypothesize that LFLI US directly and indirectly stimulates the transition of pro-inflammatory M1 macrophages to pro-healing M2 macrophages via Rac2. This project will enhance our understanding of chronic wound healing and the potential of therapeutic ultrasound to accelerate healing. Aim 1 will elucidate the direct effects of LFLI US on macrophage function and phenotype by treating inflammatory macrophages directly with LFLI US and characterizing functional changes (proliferation, migration, and phagocytosis), protein/cytokine secretion, and gene expression. Concurrently, we will validate Rac2 as the potential mechanotransduction pathway which promotes M1 to M2 macrophages transition by analyzing integrins, focal adhesion kinases, and Rac2 via confocal microscopy and RNA characterization. [Aim 2 will validate the in vitro findings from Aim 1 using our previously developed diagnostic M1/M2 score on debrided tissue from chronic wound patients treated with LFLI US.] Aim 3 will elucidate the indirect effects of LFLI US on macrophage function and phenotype via a 3D macrophage fibroblast co-culture. The results of this study will inform the optimal design of LFLI ultrasound therapy protocols, lead to a personalized, active treatment for chronic wounds, accelerate chronic wound healing, and contribute to reduced annual wound care costs.